The present invention relates, in general, to a purinergic receptor and, in particular, to a P2X7 (also designated P2Z) receptor. The invention further relates to a nucleic acid encoding the P2X7 receptor and to a method of producing P2X7 recombinantly using same. The invention also relates to a method of screening compounds for their ability to inhibit P2X7 activity and thereby for their usefulness in treating a variety of diseases/disorders, including arthritic and respiratory disorders and neurodegenerative diseases.
Cell surface receptors for ATP can be divided into metabotropic (P2Y/P2U) and ionotropic (P2X) classes. The metabotropic class belongs to the superfamily of G protein-coupled receptors, with seven transmembrane segments. The ionotropic class members (P2X1-P2X6) are ligand-gated ion channels, currently thought to be multisubunit proteins with two transmembrane domains per subunit (Buell et al, Europ. J. Neurosci. 8:2221 (1996)). P2Z receptors have been distinguished from other P2 receptors in three main ways (Buisman et al, Proc. Natl. Acad. Sci. USA 85:7988 (1988); Cockcroft et al, Nature 279:541 (1979); Steinberg et al, J. Biol. Chem. 262:3118 (1987)). First, activation of P2Z receptors leads not only to an inward ionic current, but also to cell permeabilization. Second, 3xe2x80x2-O-(4-benzoyl)benzoyl ATP (BZATP) is the most effective agonist, and ATP itself is of rather low potency. Third, responses are strongly inhibited by extracellular magnesium ions, which has been interpreted to indicate that ATP4xe2x88x92 is the active agonist (DiVirgilio, Immunol. Today 16:524 (1995)).
A seventh member of the P2X receptor family has been isolated from a rat cDNA library and, when expressed in human embryonic kidney (HEK293) cells, exhibits the above three properties (Surprenant et al, Science 272:735 (1996)). This receptor (rP2X7) thus corresponds to the P2Z receptor. rP2X7 is structurally related to other members of the P2X family but it has a longer cytoplasmic C-terminus domain (there is 35-40% amino acid identity in the region of homology, but the C-terminus is 239 amino acids long in the rP2X7 receptor compared with 27-20 amino acids in the others). The rP2X7 receptor functions both as a channel permeable to small cations and as a cytolytic pore. Brief applications of ATP (1-2s) transiently open the channel, as is the case of other P2X receptors. Repeated or prolonged applications of agonist cause cell permeabilization (which permeabilization involves the cytoplasm C-terminus); reducing the extracellular magnesium concentration potentiates this effect.
The P2Z receptor has been implicated in lysis of antigen-presenting cells by cytotoxic T lymphocytes, in the mitogenic stimulation of human T lymphocytes, as well as in the formation of multinucleated giant cells (Blanchard et al, Blood 85:3173 (1995); Falzoni et al, J. Clin. Invest. 95:1207 (1995); Baricolrdi et al, Blood 87:682 (1996)). However, the interpretation of the physiological role of P2X7 receptor has been complicated by functional differences which seem to exist between rodent and man (Hickman et al, Blood 84:2452 (1994)). The human macrophage P2X7 receptor (hP2X7) has now been cloned and its functional properties determined (Rassendren et al, J. Biol. Chem. 272:5482 (1997)).
The present invention relates to a P2X7 receptor and to a nucleic acid encoding same. The invention also relates to a method of producing P2X7 recombinantly and to a method of screening compounds for their ability to inhibit P2X7 activity. Compounds selected using the present screen can be used to treat a variety of physiological diseases and disorders.
Objects and advantages of the present invention will be clear from the description that follows.